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Behavioral consequences of methyllycaconitine in mice: a model of α7 nicotinic acetylcholine receptor deficiency
Life Sciences 74 (2004) 3133 – 3139 www.elsevier.com/locate/lifescie
Behavioral consequences of methyllycaconitine in mice: a model of a7 nicotinic acetylcholine receptor deficiency Melissa Chilton a, John Mastropaolo a, Richard B. Rosse a,b, Alan S. Bellack a,c, Stephen I. Deutsch a,b,c,* a
Mental Health Service Line, Department of Veterans Affairs Medical Center, 50 Irving Street, NW, Washington, DC 20422, USA b Department of Psychiatry, Georgetown University School of Medicine, 3800 Reservoir Road, NW, Washington, DC 20007, USA c Department of Psychiatry, University of Maryland School of Maryland, 701 West Pratt Street, Suite 388, Baltimore, MD 21201, USA Received 3 September 2003; accepted 12 November 2003
Abstract Diminished expression of the a7 nicotinic acetylcholine receptor occurs in selected brain regions of patients with schizophrenia, which may account for pathophysiological abnormalities and some of the deficits in attention and information processing. In view of this neurotransmitter receptor deficit, we wished to characterize the behavioral consequences associated with the administration of methyllycaconitine (MLA), a competitive a7 nicotinic acetylcholine receptor antagonist, in mice. In this study, we injected groups of 12 outbred NIH Swiss male mice intraperitoneally with MLA (1.0, 3.2 and 10.0 mg/kg) and its saline vehicle. Thereafter, individual mice were observed over a one-hour interval and the intensity of a variety of behaviors were rated on a 4-point scale. The observed behaviors included: gnawing/chewing, rearing, grooming, sniffing, climbing, Straub tail, locomotion and ataxia. MLA produced statistically significant changes in the following observed behaviors: rearing, sniffing, climbing, and locomotion. A profile of the behavioral changes related to MLA administration in mice could lead to the development of a screening paradigm for a7 nicotinic acetylcholine receptor agonist interventions. Ideally, an effective a7 nicotinic acetylcholine receptor agonist intervention would target domains of psychopathology, especially cognitive symptoms that contribute to the profound functional disability that is often associated with schizophrenia. D 2004 Elsevier Inc. All rights reserved. Keywords: Methyllycaconitine; Mouse; Stereotypic behavior; Schizophrenia; Cognition; Negative symptoms
* Corresponding author. Tel.: +1-202-745-8156; fax: +1-202-745-8169. E-mail address: [email protected] (S.I. Deutsch). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.11.012
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Introduction The deficient expression of a7 nicotinic acetylcholine receptors in selected areas of the brain may contribute to pathophysiological and cognitive abnormalities observable in patients with schizophrenia and their closely related biological relatives (Freedman et al., 1994; Leonard et al., 1996; Adler et al., 1998). For example, the impaired inhibition of the P50 auditory evoked potential to the second of a pair of stimuli presented 500 milliseconds apart in schizophrenia patients may be due to diminished hippocampal density of the a7 nicotinic acetylcholine receptor (Adler et al., 1998). Further, abnormal promoter variants located upstream of the gene for this receptor in the region of chromosome 15q13-14 may be a transcriptional mechanism responsible for this diminished receptor expression (Leonard et al., 2002). In addition to the deficit in P50 sensory inhibition, diminished transduction of the acetylcholine signal by the a7 nicotinic acetylcholine receptor may also be responsible for the abnormality of voluntary smooth pursuit eye movement performance in schizophrenia (Adler et al., 1998; Sherr et al., 2002). Thus, an a7 nicotinic acetylcholine receptor deficiency may play a prominent role in the pathophysiology of schizophrenia. Perhaps, this receptor deficiency manifests itself most prominently in terms of abnormalities related to attention, the cognitive processing of information, and negative symptoms. These latter abnormalities may be more predictive of poor functional outcomes, than the more striking and familiar positive symptoms of schizophrenia. Because the negative (e.g., affective flattening and social withdrawal) and cognitive (e.g., inability to sustain attention and poor planning and problem solving skills) symptom domains of psychopathology respond poorly to existing pharmacotherapy, including atypical antipsychotic medications, there is an urgent need to develop newer and more effective medications (Simosky et al., 2001; Rosse and Deutsch, 2002). Conceivably, interventions that would address a presumptive a7 nicotinic acetylcholine receptor deficiency would improve attentional and cognitive processes, and negative symptoms that impact adversely on functional outcomes (Adler et al., 1998). The existence of an animal model with deficient transduction of the acetylcholine signal by a7 nicotinic acetylcholine receptors would facilitate the identification of these candidate compounds, whose actions would target attentional, cognitive, and negative symptom domains of psychopathology linked to this receptor. Methyllycaconitine (MLA) is a selective competitive antagonist of the a7 nicotinic acetylcholine receptor; its administration should create a pharmacologically-induced state of a7 nicotinic acetylcholine receptor deficiency (Damaj et al., 1999). Theoretically, behaviors elicited by the administration of this compound in rodents would reflect the diminished tone of a7 nicotinic acetylcholine receptor-mediated neurotransmission (Stevens et al., 1998). A knockout mouse with a null mutation in the gene encoding the a7 nicotinic acetylcholine receptor subunit, termed the Acra7-deficient mouse, has been developed (Orr-Urtreger et al., 1997; Marubio and Changeux, 2000). These mice do not demonstrate a-bungarotoxin binding sites in their brains nor do they show rapidly desensitizing nicotinic currents in hippocampal neurons, consistent with a total absence of the a7 nicotinic acetylcholine receptor. Interestingly, in spite of the absence of a7 subunits, Acra7 homozygous deficient mice were surprisingly similar to their wild type littermates on a variety of baseline behaviors (Paylor et al., 1998; Marubio and Changeux, 2000). Acra7 homozygous deficient mice do not express a7-containing nicotinic acetylcholine receptors diffusely throughout their entire brain and during the entire course of their development. Of particular interest, the Acra7 homozygous deficient mice showed no decrement in the prepulse inhibition of their startle response with either auditory or tactile stimulus paradigms, compared with their wild-type littermates
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(Paylor et al., 1998). Diminished sensorimotor gating, as reflected in impaired prepulse inhibition of the startle response, is a well-known phenomenon in schizophrenia. Further, the Acra7 homozygous deficient mice showed no impairment in spatial learning or memory, as measured with the Morris water maze task (Paylor et al., 1998). Spatial learning deficits of this type are referable to the hippocampus, a region with a high density of a7 nicotinic acetylcholine receptors. These data suggest that the effects of absent expression of the a7 nicotinic acetylcholine receptor subunit in a developing brain on sensorimotor gating and spatial learning and memory are subtle at best. Alternatively, these data suggest that significant compensation for the diffuse absence of this receptor subunit during brain development can occur. It may be that the emergence of an obvious behavioral consequence of the Acra7 homozygous deficient condition requires provocation with a stressful stimulus or a pharmacological challenge. Thus, these mutant mice do not mimic the situation in schizophrenia, where diminished, but not absent, expression of normal a7-containing receptors may occur in selected brain regions (Leonard et al., 2002). In view of this, the study of behaviors elicited by MLA in normally developed mice may complement studies conducted with Acra7 homozygous deficient mice and better mimic at least some aspects of schizophrenia. In the current study, we sought to characterize the behavioral consequences of administering MLA to an outbred strain of mice. Specifically, we studied the effects of MLA on locomotor activity, ataxia and a variety of stereotypic behaviors manifested by these mice over the course of a one-hour observation period after injection. Ideally, the profile of behavioral alterations that we observed will reflect at least some aspects of a7 nicotinic acetylcholine receptor deficiency. Further, these behaviors could serve as an animal model to identify compounds that possess a7 nicotinic acetylcholine receptor agonist properties in the intact animal. These latter compounds may have therapeutic value in schizophrenia and other disorders with deficient a7 nicotinic acetylcholine receptors, such as Alzheimer’s disease and Down’s syndrome (Deutsch et al., 2002, in press). Nicotine itself was shown to transiently improve sensory gating abnormalities in schizophrenia patients and their relatives (Adler et al., 1993, 1998). Further, galantamine, a compound that is a positive allosteric modulator of nicotinic acetylcholine receptors, has been reported to improve negative symptoms in a patient with schizophrenia (Rosse and Deutsch, 2002).
Materials and methods Animals Experimentally naı¨ve outbred NIH Swiss male mice weighing 20–30 g were obtained from the National Cancer Institute (Frederick, MD U.S.A.) and housed (3 mice/cage) in hanging clear Plexiglass cages in an AALAC-approved animal facility. The animals were maintained on a 12-h light/dark cycle, with free access to food and water. On the day of testing, the mice were transported to the laboratory at least one half hour before experimentation. The animals were weighed individually before drug administration and behavioral testing. Each dose of MLA and its vehicle were tested in groups of 12 mice. The experimental protocol was approved by the Animal Studies Subcommittee and Research Committee of the Department of Veterans Affairs Medical Center in Washington, DC. All experiments were conducted in accordance with the approved protocol.
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Drugs MLA (methyllycaconitine citrate; Sigma-Aldrich Co., St. Louis, MO) was dissolved in 0.9% saline and prepared freshly on the days of the experiment. Groups of 12 mice per condition were injected intraperitoneally with MLA (1.0, 3.2 and 10.0 mg/kg) or its vehicle in a volume of 0.01 mL/g of body weight. Behavioral Ratings Individual mice randomly assigned to drug condition were injected intraperitoneally with either MLA or vehicle and placed in clear plastic cages (28 20 15 cm) that were lined with bedding and equipped with wire cage lids. Six minutes after injection, observations of the following behaviors were made for 1 minute epochs in 6 minute intervals: gnawing/chewing, rearing, grooming, sniffing, climbing, Straub tail, locomotion, and ataxia. Animals were observed for 1 hour, yielding 10 observations. The intensity of each behavior during individual observations was rated quantitatively
Fig. 1. Mean rating for observed behavior in groups of mice after intraperitoneal injection of methyllycaconitine (1.0, 3.2, 10.0 mg/kg) or its vehicle. * denotes significant difference from vehicle. + denotes significant difference from 10 mg/kg dose.
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in a standard manner: 0 (not present), 1 (sometimes present), 2 (often present), and 3 (continuously present). In each drug condition, the score for each behavior represents the mean of all observations for the group of animals. The total score was calculated by summing the mean rating of each individual behavior, excluding locomotion.
Results Data were subjected to a one-way analysis of variance (ANOVA). The ANOVA revealed significant differences for several behaviors from the observed ratings data. Subsequent to this, the data were subjected to post hoc analyses (Least Significant Differences Test) that revealed which specific doses were different. In this outbred strain of mouse, MLA produced statistically significant changes in observed behaviors, relative to vehicle. In the ANOVA, gnawing/chewing, grooming, Straub tail, and ataxia were not significant. Rearing (F(3,452) = 5.24, p < .0001), sniffing (F(3,452) = 3.27, p < .0213), climbing (F(3,452) = 6.40, p < .0004), locomotion (F(3,452) = 8.31, p < .0001), and total score (F(3,452) = 12.92, p < .0001) were found to be significant. In addition, the post hoc analyses revealed that rearing and climbing were different from vehicle for all doses of MLA. For sniffing, the 1 mg/kg dose was different from both the vehicle and 10 mg/kg dose. For locomotion and total score, the 1 mg/kg and 3.2 mg/kg doses were different from the vehicle and 10 mg/kg dose. For behaviors where significant differences were observed, the mean behavioral ratings for groups of mice given vehicle or one of the MLA doses (1, 3.2, or 10 mg/kg) are depicted in Fig. 1.
Discussion In this investigation, we examined the behavioral consequences of creating pharmacologically a condition of a7 nicotinic acetylcholine receptor deficiency in the brain of a freely moving and behaving mouse. There are compelling pathophysiological (e.g., the deficits in sensory inhibition and smooth pursuit eye movement performance) and genetic (e.g., autosomal dominant mode of inheritance of both the deficit in auditory P50 sensory gating and smooth pursuit eye movements, and an association between schizophrenia and abnormal promoter variants of the gene for the a7 nicotinic acetylcholine receptor) data supporting the existence of a condition of a7 nicotinic acetylcholine receptor deficiency in schizophrenia (Deutsch et al., in press (a),(b); Freedman et al., 1994; Leonard et al., 1996; Adler et al., 1998; Leonard et al., 2002). Further, the deficiency of the a7 nicotinic acetylcholine receptor in schizophrenia may manifest itself behaviorally as a deficit state. Many writers have both observed and commented on the high prevalence of smoking behavior in schizophrenia patients, especially those with pronounced negative symptoms (Adler et al., 1993). Conceivably, cigarette smoking represents a form of self-medication of the a7 nicotinic acetylcholine receptor deficiency in these patients. Moreover, at least some of clozapine’s therapeutic actions may be mediated by the a7 nicotinic acetylcholine receptor as its ability to improve the homologue of the human P50 sensory gating deficit in DBA/2 mice was antagonized by a-bungarotoxin, a competitive a7 nicotinic acetylcholine receptor antagonist (Simosky et al., 2003). Clozapine is an atypical antipsychotic medication that may have unique advantages over the conventional antipsychotic medications with respect to treatment-refractoriness, negative symptoms and cognition. However, in spite of the therapeutic advantages of the atypical antipsychotic medications,
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they have had only a modest impact, at best, on the deficit state of schizophrenia (Rosse and Deutsch, 2002). As noted, patients with the deficit state of schizophrenia present with symptoms reflecting attentional and cognitive disturbances and negative symptoms (Rosse and Deutsch, 2002). Negative symptoms include such items as affective flattening, social withdrawal, and lack of both motivation and energy. The mice treated with MLA showed an actual suppression in the intensity of various behaviors (i.e., rearing, sniffing, climbing and locomotion). Perhaps, the diminished intensity of expression of these behaviors due to MLA relates to the deficit state in schizophrenia (Rosse and Deutsch, 2002). In any event, these behaviors could serve as a convenient screening procedure to evaluate the ability of candidate compounds to improve transduction of the acetylcholine signal at the a7 nicotinic acetylcholine receptor.
Acknowledgements The VISN 5 Mental Illness Research, Education, and Clinical Center (MIRECC; Alan S. Bellack, Ph.D., Director) supported this work.
References Adler, L.E., Hoffer, L.D., Wiser, A., Freedman, R., 1993. Normalization of auditory physiology by cigarette smoking in schizophrenic patients. Am J Psychiatry 150, 1856 – 1861. Adler, L.E., Olincy, A., Waldo, M., Harris, J.G., Griffith, J., Stevens, K., Flach, K., Nagamoto, H., Bickford, P., Leonard, S., Freedman, R., 1998. Schizophrenia, sensory gating, and nicotine receptors. Schizophrenia Bulletin 24 (2), 189 – 202. Damaj, M.I., Glassco, W., Dukat, M., et al., 1999. Pharmacological characterization of nicotine-induced seizures in mice. JPET 291, 1284 – 1291. Deutsch, S.I., Rosse, R.B., Schwartz, B.L., Weizman, A., Chilton, M., Arnold, D.S., Mastropaolo, J., in press. Therapeutic implications of a selective a7 nicotinic receptor abnormality in schizophrenia. Israel Journal of Psychiatry. Deutsch, S.I., Rosse, R.B., Mastropaolo, J., Chilton, M., 2002. Progressive worsening of adaptive functions in Down’s Syndrome may be mediated by the complexing of soluble Ah peptides with the a7 nicotinic acetylcholine receptor: Therapeutic Implications. Clinical Neuropharmacology 26 (5), 277 – 283. Freedman, R., Adler, L.E., Bickford, P., Byerley, W., Coon, H., Cullum, C.M., Griffith, J.M., Harris, J.G., Leonard, S., Miller, C., et al., 1994. Schizophrenia and nicotinic receptors. Harvard Rev Psychiatry 2, 179 – 192. Leonard, S., Adams, C., Breese, C.R., Adler, L.E., Bickford, P., Byerley, P., Coon, H., Griffith, J.M., Miller, C., Myles-Worsley, M., et al., 1996. Nicotine receptor function in schizophrenia. Schizophrenia Bulletin 22 (3), 421 – 445. Leonard, S., Gault, J., Hopkins, J., Logel, J., Vianzon, R., Short, M., Drebing, C., Berger, R., Venn, D., Sirota, P., et al., 2002. Association of promoter variants in the nicotinic a7 acetylcholine receptor subunit gene with an inhibitory deficit found in schizophrenia. Arch Gen Psychiatry 59, 1085 – 1096. Marubio, L.M., Changeux, J.-P., 2000. Nicotinic acetylcholine receptor knockout mice as animal models for studying receptor function. Eur J Pharmacol 393, 113 – 121. Orr-Urtreger, A., Goldner, F.M., Saeki, M., Lorenzo, I., Goldberg, L., DeBiasi, M., Dani, J.A., Patrick, J.W., Beaudet, A.L., 1997. Mice deficient in the alpha7 neuronal nicotinic acetylcholine receptor lack alpha-bungarotoxin binding sites and hippocampal fast nicotinic currents. J Neurosci 17, 9165 – 9171. Paylor, R., Nguyen, M., Crawley, J.N., Patrick, J., Beaudet, A., Orr-Urtreger, A., 1998. a7 Nicotinic receptor subunits are not necessary for hippocampal-dependent learning or sensorimotor gating: a behavioral characterization of Acra7-deficient mice. Learning and Memory 5 (4), 302 – 316.
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Rosse, R.B., Deutsch, S.I., 2002. Adjuvant galantamine administration improves negative symptoms in a patient with treatment-refractory schizophrenia. Clinical Neuropharmacology 25 (5), 272 – 275. Sherr, J.D., Myers, C., Avalia, M.T., Elliott, A., Blaxton, T.A., Thaker, G.K., 2002. The effects of nicotine on specific eye tracking measures in schizophrenia. Biol Psychiatry 52, 721 – 728. Simosky, J.K., Stevens, K.E., Kem, W.R., Freedman, R., 2001. Intragastric DMXB-A, an a7 nicotinic agonist, improves deficient sensory inhibition in DBA/2 mice. Biol Psychiatry 50, 493 – 500. Simosky, J.K., Stevens, K.E., Adler, L.E., Freedman, R., 2003. Clozapine improves deficient inhibitory auditory processing in DBA/2 mice, via a nicotinic cholinergic mechanism. Psychopharmacology 165, 386 – 396. Stevens, K.E., Kem, W.R., Mahnir, V.M., Freedman, R., 1998. Selective a7 nicotinic agonists normalize inhibition of auditory response in DBA mice. Psychopharmacology 136, 320 – 327.
Behavioral consequences of methyllycaconitine in mice: a model of a7 nicotinic acetylcholine receptor deficiency Melissa Chilton a, John Mastropaolo a, Richard B. Rosse a,b, Alan S. Bellack a,c, Stephen I. Deutsch a,b,c,* a
Mental Health Service Line, Department of Veterans Affairs Medical Center, 50 Irving Street, NW, Washington, DC 20422, USA b Department of Psychiatry, Georgetown University School of Medicine, 3800 Reservoir Road, NW, Washington, DC 20007, USA c Department of Psychiatry, University of Maryland School of Maryland, 701 West Pratt Street, Suite 388, Baltimore, MD 21201, USA Received 3 September 2003; accepted 12 November 2003
Abstract Diminished expression of the a7 nicotinic acetylcholine receptor occurs in selected brain regions of patients with schizophrenia, which may account for pathophysiological abnormalities and some of the deficits in attention and information processing. In view of this neurotransmitter receptor deficit, we wished to characterize the behavioral consequences associated with the administration of methyllycaconitine (MLA), a competitive a7 nicotinic acetylcholine receptor antagonist, in mice. In this study, we injected groups of 12 outbred NIH Swiss male mice intraperitoneally with MLA (1.0, 3.2 and 10.0 mg/kg) and its saline vehicle. Thereafter, individual mice were observed over a one-hour interval and the intensity of a variety of behaviors were rated on a 4-point scale. The observed behaviors included: gnawing/chewing, rearing, grooming, sniffing, climbing, Straub tail, locomotion and ataxia. MLA produced statistically significant changes in the following observed behaviors: rearing, sniffing, climbing, and locomotion. A profile of the behavioral changes related to MLA administration in mice could lead to the development of a screening paradigm for a7 nicotinic acetylcholine receptor agonist interventions. Ideally, an effective a7 nicotinic acetylcholine receptor agonist intervention would target domains of psychopathology, especially cognitive symptoms that contribute to the profound functional disability that is often associated with schizophrenia. D 2004 Elsevier Inc. All rights reserved. Keywords: Methyllycaconitine; Mouse; Stereotypic behavior; Schizophrenia; Cognition; Negative symptoms
* Corresponding author. Tel.: +1-202-745-8156; fax: +1-202-745-8169. E-mail address: [email protected] (S.I. Deutsch). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2003.11.012
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Introduction The deficient expression of a7 nicotinic acetylcholine receptors in selected areas of the brain may contribute to pathophysiological and cognitive abnormalities observable in patients with schizophrenia and their closely related biological relatives (Freedman et al., 1994; Leonard et al., 1996; Adler et al., 1998). For example, the impaired inhibition of the P50 auditory evoked potential to the second of a pair of stimuli presented 500 milliseconds apart in schizophrenia patients may be due to diminished hippocampal density of the a7 nicotinic acetylcholine receptor (Adler et al., 1998). Further, abnormal promoter variants located upstream of the gene for this receptor in the region of chromosome 15q13-14 may be a transcriptional mechanism responsible for this diminished receptor expression (Leonard et al., 2002). In addition to the deficit in P50 sensory inhibition, diminished transduction of the acetylcholine signal by the a7 nicotinic acetylcholine receptor may also be responsible for the abnormality of voluntary smooth pursuit eye movement performance in schizophrenia (Adler et al., 1998; Sherr et al., 2002). Thus, an a7 nicotinic acetylcholine receptor deficiency may play a prominent role in the pathophysiology of schizophrenia. Perhaps, this receptor deficiency manifests itself most prominently in terms of abnormalities related to attention, the cognitive processing of information, and negative symptoms. These latter abnormalities may be more predictive of poor functional outcomes, than the more striking and familiar positive symptoms of schizophrenia. Because the negative (e.g., affective flattening and social withdrawal) and cognitive (e.g., inability to sustain attention and poor planning and problem solving skills) symptom domains of psychopathology respond poorly to existing pharmacotherapy, including atypical antipsychotic medications, there is an urgent need to develop newer and more effective medications (Simosky et al., 2001; Rosse and Deutsch, 2002). Conceivably, interventions that would address a presumptive a7 nicotinic acetylcholine receptor deficiency would improve attentional and cognitive processes, and negative symptoms that impact adversely on functional outcomes (Adler et al., 1998). The existence of an animal model with deficient transduction of the acetylcholine signal by a7 nicotinic acetylcholine receptors would facilitate the identification of these candidate compounds, whose actions would target attentional, cognitive, and negative symptom domains of psychopathology linked to this receptor. Methyllycaconitine (MLA) is a selective competitive antagonist of the a7 nicotinic acetylcholine receptor; its administration should create a pharmacologically-induced state of a7 nicotinic acetylcholine receptor deficiency (Damaj et al., 1999). Theoretically, behaviors elicited by the administration of this compound in rodents would reflect the diminished tone of a7 nicotinic acetylcholine receptor-mediated neurotransmission (Stevens et al., 1998). A knockout mouse with a null mutation in the gene encoding the a7 nicotinic acetylcholine receptor subunit, termed the Acra7-deficient mouse, has been developed (Orr-Urtreger et al., 1997; Marubio and Changeux, 2000). These mice do not demonstrate a-bungarotoxin binding sites in their brains nor do they show rapidly desensitizing nicotinic currents in hippocampal neurons, consistent with a total absence of the a7 nicotinic acetylcholine receptor. Interestingly, in spite of the absence of a7 subunits, Acra7 homozygous deficient mice were surprisingly similar to their wild type littermates on a variety of baseline behaviors (Paylor et al., 1998; Marubio and Changeux, 2000). Acra7 homozygous deficient mice do not express a7-containing nicotinic acetylcholine receptors diffusely throughout their entire brain and during the entire course of their development. Of particular interest, the Acra7 homozygous deficient mice showed no decrement in the prepulse inhibition of their startle response with either auditory or tactile stimulus paradigms, compared with their wild-type littermates
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(Paylor et al., 1998). Diminished sensorimotor gating, as reflected in impaired prepulse inhibition of the startle response, is a well-known phenomenon in schizophrenia. Further, the Acra7 homozygous deficient mice showed no impairment in spatial learning or memory, as measured with the Morris water maze task (Paylor et al., 1998). Spatial learning deficits of this type are referable to the hippocampus, a region with a high density of a7 nicotinic acetylcholine receptors. These data suggest that the effects of absent expression of the a7 nicotinic acetylcholine receptor subunit in a developing brain on sensorimotor gating and spatial learning and memory are subtle at best. Alternatively, these data suggest that significant compensation for the diffuse absence of this receptor subunit during brain development can occur. It may be that the emergence of an obvious behavioral consequence of the Acra7 homozygous deficient condition requires provocation with a stressful stimulus or a pharmacological challenge. Thus, these mutant mice do not mimic the situation in schizophrenia, where diminished, but not absent, expression of normal a7-containing receptors may occur in selected brain regions (Leonard et al., 2002). In view of this, the study of behaviors elicited by MLA in normally developed mice may complement studies conducted with Acra7 homozygous deficient mice and better mimic at least some aspects of schizophrenia. In the current study, we sought to characterize the behavioral consequences of administering MLA to an outbred strain of mice. Specifically, we studied the effects of MLA on locomotor activity, ataxia and a variety of stereotypic behaviors manifested by these mice over the course of a one-hour observation period after injection. Ideally, the profile of behavioral alterations that we observed will reflect at least some aspects of a7 nicotinic acetylcholine receptor deficiency. Further, these behaviors could serve as an animal model to identify compounds that possess a7 nicotinic acetylcholine receptor agonist properties in the intact animal. These latter compounds may have therapeutic value in schizophrenia and other disorders with deficient a7 nicotinic acetylcholine receptors, such as Alzheimer’s disease and Down’s syndrome (Deutsch et al., 2002, in press). Nicotine itself was shown to transiently improve sensory gating abnormalities in schizophrenia patients and their relatives (Adler et al., 1993, 1998). Further, galantamine, a compound that is a positive allosteric modulator of nicotinic acetylcholine receptors, has been reported to improve negative symptoms in a patient with schizophrenia (Rosse and Deutsch, 2002).
Materials and methods Animals Experimentally naı¨ve outbred NIH Swiss male mice weighing 20–30 g were obtained from the National Cancer Institute (Frederick, MD U.S.A.) and housed (3 mice/cage) in hanging clear Plexiglass cages in an AALAC-approved animal facility. The animals were maintained on a 12-h light/dark cycle, with free access to food and water. On the day of testing, the mice were transported to the laboratory at least one half hour before experimentation. The animals were weighed individually before drug administration and behavioral testing. Each dose of MLA and its vehicle were tested in groups of 12 mice. The experimental protocol was approved by the Animal Studies Subcommittee and Research Committee of the Department of Veterans Affairs Medical Center in Washington, DC. All experiments were conducted in accordance with the approved protocol.
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Drugs MLA (methyllycaconitine citrate; Sigma-Aldrich Co., St. Louis, MO) was dissolved in 0.9% saline and prepared freshly on the days of the experiment. Groups of 12 mice per condition were injected intraperitoneally with MLA (1.0, 3.2 and 10.0 mg/kg) or its vehicle in a volume of 0.01 mL/g of body weight. Behavioral Ratings Individual mice randomly assigned to drug condition were injected intraperitoneally with either MLA or vehicle and placed in clear plastic cages (28 20 15 cm) that were lined with bedding and equipped with wire cage lids. Six minutes after injection, observations of the following behaviors were made for 1 minute epochs in 6 minute intervals: gnawing/chewing, rearing, grooming, sniffing, climbing, Straub tail, locomotion, and ataxia. Animals were observed for 1 hour, yielding 10 observations. The intensity of each behavior during individual observations was rated quantitatively
Fig. 1. Mean rating for observed behavior in groups of mice after intraperitoneal injection of methyllycaconitine (1.0, 3.2, 10.0 mg/kg) or its vehicle. * denotes significant difference from vehicle. + denotes significant difference from 10 mg/kg dose.
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in a standard manner: 0 (not present), 1 (sometimes present), 2 (often present), and 3 (continuously present). In each drug condition, the score for each behavior represents the mean of all observations for the group of animals. The total score was calculated by summing the mean rating of each individual behavior, excluding locomotion.
Results Data were subjected to a one-way analysis of variance (ANOVA). The ANOVA revealed significant differences for several behaviors from the observed ratings data. Subsequent to this, the data were subjected to post hoc analyses (Least Significant Differences Test) that revealed which specific doses were different. In this outbred strain of mouse, MLA produced statistically significant changes in observed behaviors, relative to vehicle. In the ANOVA, gnawing/chewing, grooming, Straub tail, and ataxia were not significant. Rearing (F(3,452) = 5.24, p < .0001), sniffing (F(3,452) = 3.27, p < .0213), climbing (F(3,452) = 6.40, p < .0004), locomotion (F(3,452) = 8.31, p < .0001), and total score (F(3,452) = 12.92, p < .0001) were found to be significant. In addition, the post hoc analyses revealed that rearing and climbing were different from vehicle for all doses of MLA. For sniffing, the 1 mg/kg dose was different from both the vehicle and 10 mg/kg dose. For locomotion and total score, the 1 mg/kg and 3.2 mg/kg doses were different from the vehicle and 10 mg/kg dose. For behaviors where significant differences were observed, the mean behavioral ratings for groups of mice given vehicle or one of the MLA doses (1, 3.2, or 10 mg/kg) are depicted in Fig. 1.
Discussion In this investigation, we examined the behavioral consequences of creating pharmacologically a condition of a7 nicotinic acetylcholine receptor deficiency in the brain of a freely moving and behaving mouse. There are compelling pathophysiological (e.g., the deficits in sensory inhibition and smooth pursuit eye movement performance) and genetic (e.g., autosomal dominant mode of inheritance of both the deficit in auditory P50 sensory gating and smooth pursuit eye movements, and an association between schizophrenia and abnormal promoter variants of the gene for the a7 nicotinic acetylcholine receptor) data supporting the existence of a condition of a7 nicotinic acetylcholine receptor deficiency in schizophrenia (Deutsch et al., in press (a),(b); Freedman et al., 1994; Leonard et al., 1996; Adler et al., 1998; Leonard et al., 2002). Further, the deficiency of the a7 nicotinic acetylcholine receptor in schizophrenia may manifest itself behaviorally as a deficit state. Many writers have both observed and commented on the high prevalence of smoking behavior in schizophrenia patients, especially those with pronounced negative symptoms (Adler et al., 1993). Conceivably, cigarette smoking represents a form of self-medication of the a7 nicotinic acetylcholine receptor deficiency in these patients. Moreover, at least some of clozapine’s therapeutic actions may be mediated by the a7 nicotinic acetylcholine receptor as its ability to improve the homologue of the human P50 sensory gating deficit in DBA/2 mice was antagonized by a-bungarotoxin, a competitive a7 nicotinic acetylcholine receptor antagonist (Simosky et al., 2003). Clozapine is an atypical antipsychotic medication that may have unique advantages over the conventional antipsychotic medications with respect to treatment-refractoriness, negative symptoms and cognition. However, in spite of the therapeutic advantages of the atypical antipsychotic medications,
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they have had only a modest impact, at best, on the deficit state of schizophrenia (Rosse and Deutsch, 2002). As noted, patients with the deficit state of schizophrenia present with symptoms reflecting attentional and cognitive disturbances and negative symptoms (Rosse and Deutsch, 2002). Negative symptoms include such items as affective flattening, social withdrawal, and lack of both motivation and energy. The mice treated with MLA showed an actual suppression in the intensity of various behaviors (i.e., rearing, sniffing, climbing and locomotion). Perhaps, the diminished intensity of expression of these behaviors due to MLA relates to the deficit state in schizophrenia (Rosse and Deutsch, 2002). In any event, these behaviors could serve as a convenient screening procedure to evaluate the ability of candidate compounds to improve transduction of the acetylcholine signal at the a7 nicotinic acetylcholine receptor.
Acknowledgements The VISN 5 Mental Illness Research, Education, and Clinical Center (MIRECC; Alan S. Bellack, Ph.D., Director) supported this work.
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